Our crystallographic analysis of the best mutant M32 revealed that four of seven beneficial mutations (S45F, D127N, R128E, and H131I) lie within 10 Å of the active site catalytic base and are distributed on two helices in the NTD (Fig. 4A). A new clamp-like local structure formed by the S45F substitution and the other two aromatic residues, W33 and W34, appears to enhance acceptor substrate binding and facilitate catalysis by this mutant. This is consistent with a previous study showing that the S46F mutation in FutA from H. pylori 26695 increased catalytic activities (31). Moreover, crystal structures of the GT-B family enzymes, WaaG (PDB: 2IV7; GT4) (38) and GumK (PDB: 2HY7; GT70) (39), revealed similar surface-exposed aromatic hydrophobic residues (tryptophan and phenylalanine) on their N-terminal domains. As reported earlier (40), GT-B superfamily members undergo conformational changes involving movement of the NTD and CTD upon substrate binding. Notably, the three mutations D127N, R128E, and H131I clustered on a helix hinge between the NTD and CTD, likely altering the interdomain structure and influencing domain motions to providing a more reactive conformation. This likely extends to other GT-B enzymes. Through analysis of MD simulations, we proposed a dynamic model in which the NTD and CTD of GT-B enzymes undergo dynamic hinge-bending motions upon binding their respective acceptor and donor, thereby initiating domain closure (fig. S8E). Since interdomain motion appears to be essential for catalysis by GT-B type GTs, it is tempting to speculate that the hinge region could be a new hotspot for the rational design of catalytic properties into GT-B enzymes. Overall, this and other key mutated residues identified in our study highlighted important sites for engineering catalytic activities of GT-B type enzymes, which should also motivate hypothesis-driven studies into the basis of FucT substrate recognition, thereby facilitating future rational engineering efforts.
If an employer provides a vehicle that is used by an employee exclusively for business purposes and the substantiation requirements are met, there are no tax consequences or reporting required for that use. The use is treated as a working condition fringe benefit. Business use does not include commuting. Employees should maintain records to substantiate that all vehicle use was for business.
Restaurant owners and other organizations face many responsibilities regarding employment tax and reporting obligations with tipped employees. While tips are a great way for staff to make money, having tipped employees can pose a unique set of challenges for business owners, especially when it comes to payroll and payroll taxes, including how tips are taxed. Read below to find out more about such challenges, as well as effective ways to navigate through them.
You may be wondering if employers must pay taxes on tips. If you're an employer with tipped employees, your employees' tips may constitute taxable wages for payroll tax purposes. You may have more requirements come tax time, including withholding, reporting, and payment requirements. The IRS details the amount of tips required to be taxable. Tips are not taxable unless an employee makes more than a certain amount per calendar month. If your employee does make more than that amount in tips per month, you are responsible to withhold income, Social Security, and Medicare taxes on reported tips. You are also required to pay the employer's portion of Federal Insurance Contributions Act (FICA) and Federal Unemployment Tax Act (FUTA) taxes on the tips. That is why it's so important that employees accurately record and report tips to their employers. 781b155fdc